The mammalian system reacts to invading pathogens by mounting two broad defenses: the cell-mediated response and the humoral response. Viral and other intracellular infections are controlled primarily by the cell-mediated immune system. This control is achieved through recognition of foreign antigen displayed on the cell surface of an infected cell. The present invention describes a vaccine that stimulates the cell-mediated immune system and a method for immunizing mammals. The present invention also describes a method for inducing antigen-presenting cells to present specific antigens using the MHC class I processing pathway.
The cell-mediated immune system responds to endogenous antigen presented by the MHC class I processing pathway. Cells can process foreign proteins found in the cell cytosol and display relevant peptide epitopes using this processing pathway (Harding, in Cellular Proteolytic Systems, pp. 163–180 (1994); Carbone & Bevan, in Fundamental Immunology, pp. 541–567 (Paul, ed., 1989); Townsend & Bodmer, Annu. Rev. Immunol. 7: 601–624 (1989)). The MHC class I processing pathway involves digestion of the antigen by the proteasome complex and transport of the resulting peptides into the endoplasmic reticulum, where they bind to nascent MHC class I molecules (Germain & Margulies, Annu. Rev. Immunol. 11: 403–450 (1993)). Cytotoxic T lymphocytes (CTLs) specifically recognize the foreign antigen displayed by the MHC class I molecules and lyse the antigen-presenting cells. A population of memory T cells is also established that can react to presentation of the specific antigen. The cellular immune system is thus primed to swiftly respond to an intracellular infection by a pathogenic organism such as a virus.
The objective for a vaccine that stimulates the cell-mediated immune system is to deliver protein antigen to the cell cytosol for processing and subsequent presentation by MHC class I molecules. Several bacterial toxins including diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), and the pertussis adenylate cyclase (CYA) have been used in attempts to deliver peptide epitopes to the cell cytosol as internal or amino-terminal fusions (Stenmark et al., J. Cell Biol. 113: 1025–1032 (1991); Donnelly et al., Proc. Natl. Acad. Sci. U.S.A. 90: 3530–3534 (1993); Carbonetti et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 95: 295 (1995); Sebo et al., Infect. Immun. 63: 3851–3857 (1995)). These systems are restricted in their use as potential vaccines because they do not provide access to the MHC I processing pathway for antigen presentation, but instead likely operate through an alternative, less efficient pathway (see Kovacsovics-Bankowski & Rock, Science 267: 243–246 (1995); Rock, Immunology Today 17: 131–137 (1996)).
Surprisingly, the present invention provides antigen access to the MHC class I processing pathway via the anthrax binary toxin system. The Bacillus anthracis binary toxin consists of two distinct proteins (Smith & Stoner, Fed. Proc. 2: 1554–1557 (1967); Leppla, in Bacterial Toxins and Virulence Factors in Disease. Handbook of Natural Toxins, vol 8, pp. 543–572 (Moss et al., eds., 1995)). Protective antigen (PA) combines with lethal factor (LF) to make “lethal toxin” or “anthrax” toxin. (Friedlander, J. Biol. Chem. 261: 7123–7126 (1986); Leppla, Proc. Natl. Acad. Sci. U.S.A. 79, 3162–3166 (1982). In addition to lethal toxin, PA combined with edema factor (EF) makes edema toxin (Friedlander, Leppla, supra).
In this system, PA (83 kDa) binds to a protein receptor on the surface of cells. PA is then cleaved by a cellular protease (furin) and a amino-terminal 20-kDa fragment is released, leaving a 63-kDa fragment, PA63, bound to the cell (Leppla et al., in Molecular Mechanisms of Bacterial Virulence, pp. 127–139 (Kado & Crosa, eds., 1994); Klimpel et al., Proc. Natl. Acad. Sci. U.S.A. 89: 10277–10281 (1992)); Novak et al., J. Biol. Chem. 267: 17186–17193 1992)). PA63 binds to LF and the binary anthrax toxin is then endocytosed and transported into the cell. PA facilitates the delivery of LF from the endosome to the cytosol of the cell (Milne et al., J. Biol. Chem. 269: 20607–20612 (1994); Milne et al., Mol. Microbiol. 15: 661–666 (1995)). LF fusion proteins are also translocated into the cytosol by PA.
Once in the cytosol, in contrast to other binary toxin systems, the anthrax toxin and LF fusion proteins are processed by the MHC class I processing pathway. Cells treated with anthrax toxin fusion proteins are recognized and lysed by antigen specific CTLs. Dependence on processing via the MHC class I pathway was demonstrated by treating antigen-presenting cells with lactacystin, which inhibits proteasome function required for MHC class I processing. Thus, the anthrax toxin system can be used to create vaccines that efficiently stimulate the cell-mediated immune system.
In addition, binary toxins that have similar functional qualities can be used for the present invention. For example, the iota toxin of Clostridium perfringens is a binary toxin homologous to the lethal toxin of B. anthracis. Protein Ib of C. perfringens binds to protein Ia to form the toxin, and protein Ib is involved in cell surface binding and internalization of the toxin (Perelle et al., Infect. Immun., 61: 5147–5156 (1993)). The predicted amino acid sequence of Ib shows 33.9% identity with and 54.4% homology with PA (Perelle, supra).
One advantage of the anthrax system is its ability to accommodate large fusion proteins. Unlike the anthrax system, other bacterial toxin systems are limited in their capacity to deliver large protein antigen to the cell. While peptides are able to stimulate a cell-mediated immune response, whole protein antigens may be better suited for use in an effective vaccine, for two reasons. First, the epitope that is essential for protection in one genetic background may be irrelevant in another. Therefore, it is beneficial for a broadly applied T-cell vaccine to use the full length protein from which the various relevant epitopes are derived. Second, epitopes recognized by CTL are processed from the whole protein by specialized degradative machinery. In certain instances, the processing of the relevant epitopes is dependent on the flanking amino acid sequences (Del Val et al., Cell 66: 1145–1153 (1991)). Because it is currently not possible to accurately predict which epitopes are dependent on their context for proper processing, it is important to deliver the entire antigen to the cell cytosol for optimal processing and presentation. A final drawback to other bacterial toxin systems is that many individuals have already been immunized against the carrier toxin. However, anthrax toxin is not widely used for immunization.
With this invention, the efficient delivery of anthrax fusion proteins to the cytosol can be safely used as a method to intracellularly inoculate living cells with whole protein antigens. These antigens are then displayed by MHC I molecules. This system provides the basis for new, potent vaccines that target the cell-mediated immune system.